Bacterial cytoskeletal filaments: Towards a DNA segregation system for a synthetic cell

The past decades have seen the rapid development of many aspects of synthetic biology. For example, attempts to build synthetic cells under controlled conditions in the laboratory have led to significant achievements. Following a bottom-up approach, scientists aim at building a self-reproducing synthetic cell with a minimum number of biological...

Exploring Giant Unilamellar Vesicle Production for Artificial Cells – Current Challenges and Future Directions

Creating an artificial cell from the bottom up is a long-standing challenge and, while significant progress has been made, the full realization of this goal remains elusive. Arguably, one of the biggest hurdles that researchers are facing now is the assembly of different modules of cell function inside a single container. Giant unilamellar...

Rebuilding Cytokinesis One Molecule at a Time

Cells are the fundamental units of life. They make up all living things, from bacteria that live in the soil, to archea that give thermal springs their bright colors, to trees and humans. All of these cells share some common functions: they build themselves from basic building blocks, following the instructions of their genetic blueprint, and...

Complex Simplicity: Towards reconstituting Cdc42-based polarity establishment

Saccharomyces cerevisiae proliferates through budding, where a daughter cell grows by budding off one side of the mother. The first step towards budding is polarity establishment: here Cdc42 accumulates in one spot on the membrane, marking the site of bud-emergence. Cdc42 accumulation arises through at least two interconnected regulatory...

Synthetic Cell Aspirations: A Toolbox for Building a Membrane Container from the Bottom Up

Everything that we consider alive, be it plants, dogs, bacteria or humans, is composed of the same microscopic building blocks: cells. While cells between and even within these organisms can look and behave very differently, they all share the same key functionalities: they can grow, they can divide to proliferate, they can eat and metabolise to...

Actomyosin-Driven Division of a Synthetic Cell

One of the major challenges of bottom-up synthetic biology is rebuilding a minimal cell division machinery. From a reconstitution perspective, the animal cell division apparatus is mechanically the simplest and therefore attractive to rebuild. An actin-based ring produces contractile force to constrict the membrane. By contrast, microbes and...

DisGUVery: A Versatile Open-Source Software for High-Throughput Image Analysis of Giant Unilamellar Vesicles

Giant unilamellar vesicles (GUVs) are cell-sized aqueous compartments enclosed by a phospholipid bilayer. Due to their cell-mimicking properties, GUVs have become a widespread experimental tool in synthetic biology to study membrane properties and cellular processes. In stark contrast to the experimental progress, quantitative analysis of GUV...

Reconstituting microtubule-actin coordination by cytolinkers

The human body is composed of about 3−4×1013 (30-40 trillion) cells [1]. These cells are all functioning consistently, and working elegantly together, to sustain the organism. Not only humans, but all other living things on earth (from plants to parrots) are composed of cells. Cells are the smallest living building blocks of plants and animals,...

Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9 subunit (also known as SEPTIN9). Despite the established role of SEPT9 in mammalian...

On the role of microtubules in cell polarity: A reconstituted minimal system

Every living organism consists of cells. Even for the simplest single-cell organism, this cell is extremely complex. Thousands of components (such asDNA, cytoskeletal filaments, proteins, lipids, nutrients and energy) are organized both spatially and temporally to ensure proper functioning of vital cellular processes. One of those processes is...

The dynamics of microtubule stability: A reconstitution of regulated microtubule assembly under force

The microtubule cytoskeleton is an intracellular polymer network involved in cell shape, cell motility, and cell division. Microtubules are self-assembling, dynamic polymers composed of tubulin proteins that alternate between phases of growth and shrinkage, a behaviour known as “dynamic instability”. A key feature of microtubules is their...

Cytolinker Gas2L1 regulates axon morphology through microtubule-modulated actin stabilization

Crosstalk between the actin and microtubule cytoskeletons underlies cellular morphogenesis. Interactions between actin filaments and microtubules are particularly important for establishing the complex polarized morphology of neurons. Here, we characterized the neuronal function of growth arrest-specific 2-like 1 (Gas2L1), a protein that can...

Minimal in vitro systems shed light on cell polarity

Cell polarity - the morphological and functional differentiation of cellular compartments in a directional manner - is required for processes such as orientation of cell division, directed cellular growth and motility. How the interplay of components within the complexity of a cell leads to cell polarity is still heavily debated. In this...

A minimal system to establish microtubule-based cell polarity in vitro

In this thesis we took the challenge to in vitro reconstitute a minimal phenomenon essential for live: Cell polarity. This is an ubiquitous phenomenon that allows cells to define a direction for migration, growth or division. Our study focussed on microtubulebased establishment of polarity taking S. pombe as a model organism. In this rod-shaped...

Understanding force-generating microtubule systems through in vitro reconstitution

Microtubules switch between growing and shrinking states, a feature known as dynamic instability. The biochemical parameters underlying dynamic instability are modulated by a wide variety of microtubule-associated proteins that enable the strict control of microtubule dynamics in cells. The forces generated by controlled growth and shrinkage...

Reconstitution of sensitive clays

In this thesis a method is developed to regain the geotechnical behaviour of undisturbed quick clay by reconstitution of remoulded natural quick clay samples. Quick clay is characterised by a high sensitivity; the ratio between the undisturbed and remoulded strength is very high. Because of the high water content they behave fluid-like after...